Multiple Inherited Thrombophilic Gene Polymorphisms in Spontaneous Abortions in Turkish Population.

The aim of this study was to investigate the possible role of multiple inherited thrombophilic gene variations in women with unexplained spontaneous abortions. For this purpose, the Factor V Leiden (FVL) (rs6025), Prothrombin G20210A (rs1799963), MTHFR C677T (rs1801133), PAI-1 4G/5G (rs1799889), ACE I/D (rs1799752), eNOS E298D (rs1799983), and Apo E E2/E3/E4 (rs429358) polymorphisms were genotyped and correlated in spontaneously aborted fetal materials, their mothers and fertile women. Twenty three abortion materials, 22 women with ≥1 unexplained fetal loss, and 22 control subjects with at least two healthy term infants as a control group were studied. Target SNPs for each gene were analyzed by real time-PCR technique after genomic DNA isolation from maternal blood-EDTA, control group blood-EDTA and spontaneously aborted fetal tissues. Some cases had a single thrombophilic polymorphism, but the rest of the patients and fetal materials had combined thrombophilic polymorphisms. The PAI-1 4G/5G+4G/4G (P= 0.0017), 4G/4G (P= 0.0253), eNOS 894GT+894TT (P=0.0011) genotypes and T allele (P=0.0185), Apo E E3/E4+E3/E2+E2/E4 (P<0.0001) genotypes, E2 (P<0.0001) and E4 (P<0.0001) alleles were higher in spontaneously aborted fetal materials when compared to their mothers and control group. The Factor V Leiden rs6025, Prothrombin G20210A, MTHFR C677T, ACE I/D genotypes were different for each group but not statistically significant due to relatively small size of the samples (P>0.05). Our results indicated that combined thrombophilic gene variations may be associated with increased risk for spontaneous abortions and results need to be confirmed by larger sample size.

In this study, we aimed to compare the thrombophilic polymorphisms in Factor V Leiden (rs1799983), and Apo E E2/E3/E4 (rs429358) in spontaneously aborted fetal materials with their mothers and fertile cases. This is the first study about thrombophilia polymorphisms in spontaneously aborted fetal materials.

Patients and clinical diagnosis
Twenty-three abortion materials, their twentytwo mothers (one of the mothers had two abortions) and twenty-two fertile women as control group were included in this study. Abortion materials were obtained from only spontaneous abortions.
Maternally contaminated abortion materials (twenty-seven materials) were excluded from the study (fifty abortion materials were taken for this study

Statistical analysis
Statistical analysis has been done with medcalc statistical program. Chi-square test was used to analyze the differences between the abortion materials, mothers and fertile women as control group. Genotype percentages and allele frequencies were compared between aborted materials group-mothers group and aborted materials group-control group. Odds ratio (OR) and P-values were used to estimate the risk for the polymorphisms in the groups.

Results
Peripheral blood-EDTA samples from mothers and control cases (fertile women) and tissue samples from aborted materials were examined for genotyping in the current study. Mean age of the cases and the weeks of abortions are provided. The mean age of the mothers were 29.6 (21-40) years and the mean age of fertile women were 37.2 (30-46) years. The mean abortion week was 9.4 (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) weeks, twenty abortions were in first trimester, three abortions were in second trimester. There was no other diseases in the groups.  but not with recurrent pregnancy loss (12). In another study, FVL and MTHFR polymorphisms were found as not associated with recurrent pregnancy loss or pre-eclampsia (13).Generally, studies belong to different countries and races, so that polymorphism results may differ. For example, a study from China reported that there was a significant association between MTHFR C677T and unexplained recurrent pregnancy loss (14). A study planned with couples, reported that MTHFR C677T polymorphism is a risk factor for RPL (15 reported that PAI-1 (-675) 4G/4G homozygous genotype was associated with increased risk of RPL independently from the antiphospholipid antibodies (19). In a different study, it was reported that ACE I/D, PAI-1 4G/5G and NOS3 4a/4b were not associated with first trimester recurrent miscarriage (20). Agarwal et.al. reported that there was no association between Apo E gene polymorphisms and RPL (21). In our results Apo E2 and E4 alleles and genotypes with these alleles were significant comparing with E3 allele and E3/E3 genotype between fetus and mother groups. Fetal Apo E genotype is found as associated with spontaneous abortion.
In conclusion, spontaneous abortion risk is related with PAI-1 4G/5G, eNOS E298D and Apo E E2/E3/E4 polymorphisms for fetal genotypes. If fetal genotypes are effective comparing with mother genotypes, this means father genotypes are also effective for pregnancy losses. Our results indicated that combined thrombophilic gene mutations may be associated with increased risk for spontaneous abortions. There is a need for larger studies to explore the effect of thrombophilic factors in family members for pregnancy losses.